Ion detection using a pillar chip

ABSTRACT

The present invention provides methods and assemblies for ion detection of samples using a chip with elevated sample zones. The elevated sample zones provide a number of ion detection advantages over chips with non-elevated sample zones. Embodiments of the invention have a number of applications in drug discovery, environmental analyses for tracking and the identification of contaminants, target discovery and/or validation as well as in diagnostics in a clinical setting for staging or disease progression. In addition, the invention may also be used with research and clinical microarray systems and devices.

BACKGROUND OF THE INVENTION

the discovery of new drugs, potential drug candidates are generated byidentifying chemical compounds with desirable properties. Thesecompounds are sometimes referred to as “lead compounds”. Once a leadcompound is discovered, variants of the lead compound can be created andevaluated as potential drug candidates.

In order to reduce the time associated with discovering useful drugcandidates, high throughput screening (HTS) methods are replacingconventional lead compound identification methods. High throughputscreening methods use libraries containing large numbers of potentiallydesirable compounds. The compounds in the library are numerous and maybe made by combinatorial chemistry processes. In a HTS process, thecompounds are screened in one or more assays to identify those librarymembers (particular chemical species or subclasses) that display adesired characteristic activity. The compounds thus identified can serveas conventional “lead compounds” or they can be therapeutic.

Conventional HTS processes use multi-well plates having many wells. Forexample, a typical multi-well plate may have 96 wells. Each of the wellsmay contain a different liquid sample to be analyzed. Using a multi-wellplate, a number of different liquid samples may be analyzedsubstantially simultaneously.

It is desirable to reduce the volume of the wells in a multi-well plateto increase the density of the wells on the plate. By doing so, morewells can be present on the plate and more reactions can be analyzedsubstantially simultaneously. Also, as the volumes of the wells arereduced, the liquid sample volumes are reduced. Reducing the liquidsample volumes reduces the amount of reagents needed in the HTS process.By reducing the amount of reagents used, the costs of the HTS processcan be reduced. Also, liquid samples such as samples of biologicalfluids (e.g., blood) are not always easy to obtain. It is desirable tominimize the amount of sample in an assay in the event that littlesample is available.

While it is desirable to increase the density of the wells in amulti-well plate, the density of the wells is limited by the presence ofthe rims on the wells. The rims could be removed to permit the samplezones to be closer together and thus increase the density of the samplezones. However, by removing the rims, no physical barrier would bepresent between adjacent sample zones. This increases the likelihoodthat liquid samples on adjacent sample zones could intermix andcontaminate each other.

Also, reducing the liquid sample volumes can be problematic. Decreasingthe size of assays to volumes smaller than I microliter substantiallyincreases the surface-to-volume ratio. Increasing the surface-to-volumeratio increases the likelihood that analytes or capture agents in theliquid sample will be altered, thus affecting any analysis or reactionusing the analyte or capture agents. For example, proteins in a liquidsample are prone to denature at liquid/solid and liquid/air interfaces.When a liquid sample containing proteins is formed into a droplet, thedroplet can have a high surface area relative to the amount of proteinsin the droplet. If the proteins in the liquid sample come into contactwith the liquid/air interface, the proteins may denature and becomeinactive. Furthermore, when the surface-to-volume ratio of a liquidsample increases, the likelihood that the liquid sample will evaporatealso increases. Liquids with submicroliter volumes tend to evaporaterapidly when in contact with air. For example, many submicrolitervolumes of liquid can evaporate within seconds to a few minutes. Thismakes it difficult to analyze or process such liquids. In addition, ifthe liquid samples contain proteins, the evaporation of the liquidcomponents of the liquid samples can adversely affect (e.g., denature)the proteins.

Chips having elevated sample zones solve many of the problems associatedwith the use of multi-well plated for HTS processes (see U.S. patentapplication Ser. No. 09/792,335, filed Feb. 23, 2001, entitled “ChipsHaving Elevated Sample Surfaces”).

The identification of library members in HTS requires a fast andefficient method of analysis. The paucity of efficient library compoundidentification techniques remains a serious limitation for routine useof HTS processes such as protein analysis. Mass spectrometry is one suchtechnique that can potentially be used for various HTS processes such asprotein analysis. Mass spectrometry combines high sensitivity,selectivity and specificity with speed of analysis. For example, acomplete mass spectrum can be recorded on a microsecond timescale.

Thus, there is a need in the art to adapt highly sensitive massspectrometry techniques to high throughput screening methodologies suchas protein analysis. Embodiments of the invention address, for example,these and other problems.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention provide methods and assemblies for iondetection of samples using a chip with elevated sample zones. Theelevated sample zones provide a number of ion detection advantages overchips with non-elevated sample zones, such as improved desorption andionization of samples, a decrease in desorption of contaminants fromnon-sample areas, and improved electric field configurations.Embodiments of the invention have a number of applications in drugdiscovery, environmental analyses for tracking and the identification ofcontaminants, target discovery and/or validation as well as indiagnostics in a clinical setting for staging or disease progression. Inaddition, the invention may also be used with research and clinicalmicroarray systems and devices.

One embodiment is directed to a method of analyzing a sample comprisingdesorbing a sample from a chip to produce a desorbed ion sample anddetecting the desorbed ion sample. The chip comprises a base having asurface and one or more structures protruding above the surface of thebase. Each structure comprises a pillar and a sample zone. The samplezone comprises a support material and the sample to be analyzed.

Another embodiment is directed to an analytical assembly a chip and aconductive element. The chip comprises a base having a surface and oneor more structures protruding above the surface of the base. Eachstructure comprises a pillar and a sample zone. The addition, the samplezone comprises a support material. The conductive element comprises anaperture of sufficient proportion to allow passage of a molecular ionand is adapted to be at a different electrical potential than the base.

Another embodiment is directed to a mass spectrometer apparatuscomprising an analytical assembly, an ionization source to ionize thesample, and an ion detector for detecting an ion desorbed from thesample zone. The analytical assembly comprises a chip and a conductiveelement. The chip comprises a base having a surface and one or morestructures protruding above the surface of the base. Each structurecomprises a pillar and a sample zone. The addition, the sample zonecomprises a support material. The conductive element comprises anaperture of sufficient proportion to allow passage of a molecular ionand is adapted to be at a different electrical potential than the base.

These and other embodiments are described on further detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates laser desorption of a sample from the sample zone

FIG. 2 illustrates a cross-sectional view of an exemplary chip.

FIGS. 3(a)-3(b) illustrates cross sectional views of exemplary samplezones.

FIGS. 4 illustrates an exemplary laser desorption of a sample from thesample zone through the pillar.

FIG. 5 illustrates an exemplary ion detection of a desorbed ion sampleusing a mass spectrometer.

FIG. 6 illustrates an example of allowing the desorbed ion sample topass through an aperture of a conductive element.

FIG. 7 illustrates an exemplary passing of a laser through a conductiveelement.

FIG. 8(a)-(b) shows exemplary surface coatings that coat the supportmaterial.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention may be used in any number of differentfields. For example, embodiments of the invention may be used inpharmaceutical applications such as proteomic (or the like) studies fortarget discovery and/or validation as well as in diagnostics in aclinical setting for staging or disease progression. Also, embodimentsof the invention may be used in environmental analyses for tracking andthe identification of contaminants. In academic research environments,embodiments of the invention may be used in biological or medicalresearch. Embodiments of the invention may also be used with researchand clinical microarray systems and devices.

In embodiments of the invention, events such as binding, bindinginhibition, reacting, or catalysis between two or more components can beanalyzed. For example, the interaction between an analyte in a liquidsample and a binding agent bound to a sample zone on a pillar may beanalyzed using embodiments of the invention. More specifically,interactions between the following components may be analyzed usingembodiments of the invention: antibody/antigen, antibody/hapten,enzyme/substrate, carrier protein/substrate, lectin/carbohydrate,receptor/hormone, receptor/effector, protein/DNA, protein/RNA,repressor/inducer, DNA/DNA and the like.

In one embodiment, the present invention provides a method of analyzinga sample comprising desorbing a sample from a chip to produce a desorbedion sample and detecting the desorbed ion sample. The chip comprises abase having a surface and one or more structures protruding above thesurface of the base. Each structure comprises a pillar and a samplezone. The sample zone comprises a support material and the sample to beanalyzed. Once the desorbed ion sample is detected, it can be analyzedto determine its physical properties, chemical properties, quantity,etc.

In another embodiment, the present invention provides an analyticalassembly comprising a chip and a conductive element. The chip comprisesa base having a surface and one or more structures protruding above thesurface of the base. Each structure comprises a pillar and a samplezone. In addition, the sample zone comprises a support material. Theconductive element comprises an aperture of sufficient proportion toallow passage of a molecular ion and is adapted to be at a differentelectrical potential than the base.

In an exemplary embodiment, desorption of the sample is accomplished bydirecting radiation to the sample zone. Typically, a laser desorptiontechnique is used wherein the desorbing radiation is pulsed laserradiation. FIG. 1 illustrates an exemplary laser desorption technique.The laser radiation source 10 directs radiation 150 to the sample zone 6resulting in desorption of the sample from the sample zone to from adesorbed ion sample 11.

The Chip

The chip comprises a base including a base surface and one or morestructures comprising a pillar and a sample zone. The one or morestructures are typically in an array on the base of the chip. Eachstructure includes a sample zone that is elevated with respect to thebase of the chip.

In an exemplary embodiment, the structures are arranged in an arrayformat. Structure arrays of the current invention may be regular orirregular. For example, the array may have even rows of structuresforming a regular array of pillars. The density of the structures in thearray may vary. For example, the density of the structures may be about25 pillars per square centimeter or greater (e.g., 10,000 or 100,000 percm² or greater). Although the chips may have any suitable number ofstructures, in some embodiments, the number of structures per chip maybe greater than 10, 100, or 1000. The structures pitch (i.e., thecenter-to-center distance between adjacent structures) may be 500micrometers or less (e.g., 150 micrometers).

Each sample zone may be adapted to receive a sample to be processed oranalyzed while the sample is in the sample zone. The sample may be orinclude a component that is to be bound, adsorbed, absorbed, reacted,etc. within the sample zone. For example, the sample can be a liquidcontaining analytes and a liquid medium. In another example, the samplemay be the analytes themselves. Because a number of sample zones are oneach chip, many samples may be processed or analyzed in parallel inembodiments of the invention.

Adjacent sample zones are separated by a depression that is formed byadjacent pillars and the base surface. In some embodiments, the pillarsmay have one or more channels that surround, wholly or in part, one ormore pillars on the base. Examples of such channels are discussed inU.S. patent application Ser. No. 09/353,554 which is assigned to thesame assignee as the present application and which is hereinincorporated by reference in its entirety for all purposes.

Elevating the sample zone with the pillar with respect to the chip baseprovides a number of advantages. For example, by elevating the samplezone, potential liquid cross-contamination between the liquid samples onadjacent structures is minimized. A liquid sample within a sample zonedoes not easily flow to an adjacent sample zone because the sample zonesare separated by a depression. In some embodiments, cross-contaminationbetween samples on adjacent sample zones is reduced even though rims arenot present to confine a liquid sample to a sample zone. Since rims neednot be present to confine the samples to their respective sample zones,the spacing between adjacent sample zones can be reduced, thusincreasing the density of the sample zones. As a result, more liquidsamples may be processed and/or analyzed per chip than in conventionalmethods. In addition, small liquid sample volumes can be used inembodiments of the invention so that the amount of reagents used is alsodecreased, thus resulting in lower costs.

FIG. 2 illustrates an exemplary embodiment of a chip 1. The chip 1 inFIG. 2 comprises a base 2 and a surface of the base 3. The chip 1 hasthree structures 4(a), 4(b), 4(c). The structures 4(a), 4(b), 4(c)protrude above the surface of the base 2. Each structure 4(a), 4(b),4(c) comprises a pillar 5(a), 5(b), 5(c) and a sample zone 6(a), 6(b),6(c). Only three structures are shown for ease of illustration. Otherchip embodiments could have tens or hundreds of such structures.

Each of the structures may be oriented substantially perpendicular withrespect to the base 2. Each of the structures 4(a)-4(c) include a sidesurface. The side surfaces of the structures 4(a)-4(c) can definerespective sample zones 6(a)-6(c). The sample zones 6(a)-6(c) maycoincide with the top portions of the pillars 5(a)-5(c) and are elevatedwith respect to the base surface 3 of the chip 1. The base surface 3 andthe sample zones 6(a)-6(c) may have the same or different coatings orproperties.

Each sample zone comprises a support material and a sample. As usedherein, a “sample zone” refers to a zone of a structure that includes asample. A sample zone may or may not include a support material. Forexample, in one embodiment, the sample zone may include only a sample(e.g., proteins in a liquid medium) on top of a solid layer of supportmaterial on a pillar. In another embodiment, the sample zone may includea sample that is 10 impregnated in a porous support material. The poroussupport material may be separate and distinct from the pillar or may beintegral with the pillar. For instance, in the latter case, the entirepillar may be a porous material and the sample may only impregnate thetop portion of the porous pillar.

As used herein, a “support material” is a material that supports asample. The support material can be porous or solid.

FIG. 3(a) illustrates an exemplary embodiment of the sample zone 6comprising a sample 8 positioned above the support material 9. In thisexample, the support material can be a portion of the pillar 5 or can beone or more layers on the pillar 5.

FIG. 3(b) illustrates another exemplary embodiment, wherein the sample 8is present throughout the support material 9. Typically, where thesample is present throughout the support material, the support materialis porous.

The sample zones may have any suitable geometry. The geometry of thesample zone may be the same or different than the pillar of thestructure. For example, the sample zone may be circular while the pillaris square or octahedral. Each sample zone may have any suitable widthincluding a width of less than about 0.5 mm (e.g., 100 micrometers orless). The height of the sample zone may be greater than 100 micrometersor less than about 10 nanometers.

The sample zone may include one or more layers of material and/orsupport material. In some embodiments, the sample zone may be inherentlyhydrophilic or rendered hydrophilic, which are less likely to adverselyaffect proteins that may be at the top regions of the structures.

In some embodiment, the sample zone may comprises a first layer and asecond layer, wherein the second layer is on top of the first layer. Thefirst and/or the second layer may comprise the sample. The first and thesecond layers may comprise any suitable material having any suitablethickness. The first and the second layers can comprise inorganicmaterials and may comprise at least one of a metal or an oxide such as ametal oxide. The selection of the material used in, for example, thesecond layer (or for any other layer or at the top of the pillar) maydepend on the molecules that are to be bound to the second layer. Forexample, metals such as platinum, gold, and silver may be suitable foruse with linking agents such as sulfur containing linking agents (e.g.,alkanethiols or disulfide linking agents), while oxides such as siliconoxide or titanium oxide are suitable for use with linking agents such assilane-based linking agents. The linking agents can be used to coupleentities such as binding agents to the pillars.

Illustratively, the first layer may comprise an adhesion metal such astitanium and may be less than about 5 nanometers thick. The second layer29 may comprise a noble metal such as gold and may be about 100 to about200 nanometers thick. In another embodiment, the first layer 26 maycomprise an oxide such as silicon oxide or titanium oxide, while thesecond layer 29 may comprise a metal (e.g., noble metals) such as goldor silver. The sample zone may have more or less then two layers (e.g.,one layer) on them. Moreover, although the first and the second layersare described as having specific materials, it is understood that thefirst and the second layers may have any suitable combination ofmaterials.

The layers in the sample zone may be deposited using any suitableprocess. For example, the previously described layers may be depositedusing processes such as electron beam or thermal beam evaporation,chemical vapor deposition, sputtering, or any other technique known inthe art.

In some embodiments, the side or portion of the side surfaces of thepillars may be provided with the same selected properties as the samplezone, or different selected properties from the sample zone. In oneexemplary embodiment, the side surfaces of a pillar of a chip comprisesthe support material of the sample zone. In another exemplaryembodiment, side surfaces of a pillar of a chip is rendered hydrophobicwhile the sample zone of the pillar is hydrophilic. The hydrophilicsample zone of a pillar attracts the liquid samples, while thehydrophobic side surfaces of the pillar inhibit the liquid samples fromflowing down the sides of the pillars. Accordingly, in some embodiments,a liquid sample may be confined to the sample zone of a pillar without awell rim. Consequently, in embodiments of the invention,cross-contamination between adjacent sample zones may be minimized whileincreasing the density of the sample zones.

The base of the chip may have any suitable characteristics. Forinstance, the base of the chip can have any suitable lateral dimensions.For example, in some embodiments, the base can have lateral dimensionsless than about 2 square inches. In other embodiments, the base can havelateral dimensions greater than 2 square inches. The base surface may begenerally planar. However, in some embodiments, the base may have a nonplanar surface. For example, the base may have one or more troughs. Thestructures containing the sample zones and the pillars may be in thetrough. Any suitable material may be used in the base. Suitablematerials include glass, silicon, or polymeric materials. Preferably,the base comprises a micromachinable material such as silicon.

The pillars may have any suitable geometry. For example, thecross-sections (e.g., along a radius or width) of the pillars may becircular or polygonal. Each of the pillars may also be elongated. Whilethe degree of elongation may vary, in some embodiments, the pillars mayhave an aspect ratio of greater than about 0.25 or more (e.g., 0.25 to40). In other embodiments, the aspect ratio of the pillars may be about1.0 or more. The aspect ratio may be defined as the ratio of the heightH of each pillar to the smallest width W of the pillar. Preferably, theheight of each pillar may be greater than about 1 micron. For example,the height of each pillar may range from about 1 to 10 micrometers, orfrom about 10 to about 200 micrometers. Each pillar may have anysuitable width including a width of less than about 0.5 mm (e.g., 100micrometers or less). A variety of shapes and sizes of structures andpillars are useful in the current invention. Structure and pillar sizesand shapes are described in U.S. patent application Ser. No. 09/792,335,U.S. patent application Ser. No. 10/208,381, U.S. Patent Application No.60/184,381, U.S. Patent Application No. 60/225,999, and U.S. Pat. No.6,454,924, which is assigned to the same assignee as the presentapplication and which is herein incorporated by reference in itsentirety for all purposes.

The pillars of the chip may be fabricated in any suitable manner andusing any suitable material. For example, an embossing, etching or amolding process may be used to form the pillars on the base of the chip.For example, a silicon substrate can be patterned with photoresist wherethe top surfaces of the pillars are to be formed. An etching processsuch as a deep reactive ion etch may then be performed to etch deepprofiles in the silicon substrate and to form a plurality of pillars.Side profiles of the pillars may be modified by adjusting processparameters such as the ion energy used in a reactive ion etch process.If desired, the side surfaces of the formed pillars may be coated withmaterial such as a hydrophobic material while the top surfaces of thepillars are covered with photoresist. After coating, the photoresist maybe removed from the top surfaces of the pillars. Other processes forfabricating pillars known in the semiconductor and MEMS(microelectromechanical systems) industries are also useful in thepresent invention.

Desorption and Ionization

The method of the present aspect involves desorbing the sample from thesample zone to produce a desorbed ion sample. Desorption is the processof removing the sample from the sample zone. To produce a desorbed ionsample, the sample is desorbed and ionized.

In an exemplary embodiment, desorption of the sample is accomplished bydirecting radiation to the sample zone. Typically, a laser desorptiontechnique is used wherein the desorbing radiation is pulsed laserradiation. FIG. 3(a) illustrates an exemplary laser desorptiontechnique. The laser radiation source 10 directs radiation 150 to thesample zone 6 resulting in desorption of the sample from the sample zoneto from a desorbed ion sample 11.

In another exemplary embodiment, the laser radiation 150 is directed toa sample zone from below the chip through the pillar 5 (see FIG. 4). Inthis embodiment, the pillar is typically comprised of materials thatabsorb little or no light radiation.

In another embodiment, the laser desorption technique is a matrixassisted laser desorption technique (MALDI). In this MALDI embodiment,the laser is directed to the support material 7 within the sample zone6. The support material typically comprises a chemical matrix in theMALDI embodiment. Without being limited by any particular theory, thechemical matrix absorbs the laser light energy and produces a plasmathat results in desorption and ionization of the sample (see Barber etal., Nature 293: 270-275 (1981); Karas et al., Anal. Chem. 60: 2299-2301(1988); Macfarlane et al, Science 191: 920-925 (1976); Hillenkamp etal., Anal. Chem. 63: A1193-A1202 (1991)). Thus, in another embodiment,the support material is capable of transferring energy to the sampleafter receiving radiation.

In one embodiment, the sample zone comprises a support material thatreceives radiation. In another embodiment, the pillar and/or the baseadditionally comprise a support material that receives radiation.

In another embodiment, the support material is porous. Typically, theporous support material comprises a chemical matrix. In anotherembodiment, the support material is conducting or semiconducting. Avariety of chemical matrices are useful in the present invention.Chemical matrices should be capable of transferring energy to the sampleafter receiving laser radiation. Suitable chemical matrices includeporous silicon matrices. See Amato et al., Optoelectronic Properties ofSemiconductors and Superlattices, 3-52 (1997). Porous silicon surfacesare strong absorbers of ultraviolet radiation. The preparation andphotoluminescent nature of porous silicon surfaces is described byCullis et al., Appl. Phys. Lett. 82: 909, 911-912 (1997). Cullis et al,also describe and review other photoluminescent porous semiconductorssuitable for the approach described herein that exhibit the necessarystrong absorption, including SiC, GaP, Si_(1-x), Ge_(x), Ge, and GaAs,and also InP that exhibits weak photoluminescence. Porosity properties,preparation, and modification of porous silicon surfaces for use inMALDI is desorbed in U.S. Pat. No. 6,288,390, which is hereinincorporated by reference.

Other useful matrices include SiC, GaP, Si_(1-x), Ge_(x), Ge, GaAs, InP(see Cullis et al., Appl. Phys. Lett. 82: 909, 911-912 (1997)), Group IVsemiconductors (for example diamond and α-San), I-VII semiconductors(for example CuF, CuCl, CuBr, CuI, AgBr, and AgI), Group II-VIsemiconductors (for example BeO, BeS, BeSe, BeTe, BePo, MgTe, ZnO, ZnS,ZnSe, ZnTe, ZnPo, CdS, CdSe, CdTe, CdPo, HgS, HgSe, and HgTe), GroupIII-V semiconductors (for example BN, BP, BAs, AIN, AlP, AlAs, AlSb,GaN, GaP, GaSb, InN, InAs, InSb), Sphaelerite Structure Semiconductors(for example MnS, MnSe, β-SiC, Ga₂Te₃, In₂Te₃, MgGeP₂, ZnSnP₂, andZnSnAs₂), Wurtzite Structure Compounds (for example NaS, MnSe, SiC,MnTe, Al₂S₃, and Al₂Se₃), I-II-VI₂ semiconductors (for example CuAlS₂,CuAlSe₂, CuAlTe₂, CuGaS₂, CuGaSe₂, CuGaTe₂, CuInS₂, CuInSe₂, CuInTe₂,CuTlS₂, CuTlSe₂, CuFeS₂, CuFeSe₂, CuLaS₂, AgAS₂, AgAlSe₂, AgAlTe₂,AgGaS₂, AgGaSe₂, AgGaTe₂, AgInS₂, AgInSe₂, AgInTe₂, AgFeS₂), and Al₂O₃.Other conducting or semiconducting materials, such as metals andsemimetals, which absorb light and are capable of transmitting the lightenergy to an analyte to ionize it are within the scope of the inventionas well.

In another exemplary embodiment, the sample 8 is desorbed from thesample zone 6 by applying radiation directly to the sample. Typically,the radiation is light radiation, such as a laser radiation. Typically,the radiation desorbs the sample from the sample zone and ionizes thesample thereby producing a desorbed ion sample 9. For examples of directdesorption ionization see: Zenobi et al., Chimia 51: 801-803 (1997);Zhan, et al., J. Am. Soc. Mass Spec. 8: 525-531 (1997); Hrubowchak etal., Anal. Chem. 63: 1947-1953 (1991); Varakin et al., High EnergyChemistry 28: 406-411 (1994); Wang et al., Appl. Surf. Sci. 93: 205-210(1996); and Posthumus et al., Anal. Chem. 50: 985-991 (1978).

In another exemplary embodiment, the sample is desorbed using a particlebombardment technique. Particle bombardment techniques use a particlebeam directed to the sample zone to desorb the sample. The sample isdesorbed in the form of ions, fragments, or a combination thereof In oneembodiment, a fast atom bombardment technique is used to desorb thesample. In the FAB embodiment, a fast atom beam (e.g. 6 keV xenon atoms)is directed to a liquid matrix in which the sample is dissolved. Usefulliquid matrices include glycerol, thioglycerol, m-nitrobenzyl alcohol,or dithanolamine. In another embodiment, an ion beam (e.g. cesium ions)is used to desorb the sample and produce a desorbed ion sample.

In another exemplary embodiment, the sample is desorbed using a fielddesorption technique. Typically, the sample zone comprises an emitter onwhich the sample is deposited. A current is passed through the emitterand the sample is desorbed by evaporation into the gas phase to form agas phase desorbed sample. The gas phase desorbed sample is typicallyionized using a field ionization technique. An electric field at the tipof the emitter allows ionization of the gas phase desorbed sample byelectron tunneling. Emitters useful in the current invention includecarbon emitters and silicon emitters.

In another exemplary embodiment, the sample is thermally desorbed fromthe sample zone to produce a gas phase desorbed sample. The gas phasedesorbed sample is then ionized. Useful methods of ionizing a gas phasedesorbed sample include electron ionization, chemical ionization,desorption chemical ionization and negative-ion chemical ionization.

In another exemplary embodiment, the sample is thermally desorbed fromthe sample zone to produce a solution phase desorbed sample. Thesolution phase desorbed sample is then ionized. Useful method ofionizing a liquid sample include electrospray ionization and atmosphericpressure chemical ionization.

In another exemplary embodiment, electrospray ionization is performedupon a liquid sample wherein the liquid sample is desorbed from thesample zone with a liquid force. Typically, the liquid force is asolvent flowing through a pillar channel located in the pillar. Thesolvent flows from the pillar toward the sample zone (for moreinformation on channels in a pillar, see U.S. Pat. No. 6,454,924 whichherein incorporated by reference in its entirety for all purposes). Toionize the sample, a voltage may be applied to the sample zone.Elevating the sample zone with respect to the chip base provides anadvantage in performing electrospray ionization.

Elevated sample zones of the present invention provide a number ofadvantages over non-elevated sample zones. For example, elevated samplezones provide increased sample concentrations. Mass spectrometrictechniques, such as MALDI mass spectrometry, require high concentrationsof sample in order to obtain accurate results. Application of a liquidsample to a non-elevated sample zone results in a diffuse pool becausethere is no barrier to prevent the liquid from dispersing. By contrast,an elevated sample zone provides a coherent volume physically separatedfrom the base by the pillar. Thus, the elevated sample zone preventsdispersion of the sample resulting in higher concentration and improvedresults using mass spectrometry.

Another advantage of elevated sample zones is improved desorption andionization. The physical separation of the elevated sample zone from thenon-sample zones by the pillar results in sample droplets with highersurface tension. The high surface tension is desirable in forming aTaylor cone. A Taylor cone forms when an accumulation of charge causesdestabilization of the liquid surface to a point where the mutualrepulsion between charged species overcomes the surface tension (theRayleigh limit), thereby forming solvent-free ions. Thus, by improvingTaylor cone formation, the elevated sample zone provides improveddesorption and ionization.

Elevation of the sample zone also provides a greater degree ofseparation between the sample zone and the non-sample zones of the chip.The elevated sample zone provides three-dimensional separation ascompared to the two-dimensional separation of non-elevated sample zones.The higher degree of separation enables facile application of radiationto the sample. In addition, the higher degree of separation decreasesthe receipt of radiation in non-sample zones, thus decreasing desorptionof contaminating materials.

In addition, the elevated sample zone allows the electric field strengthto be varied between the base and the elevated sample zone. Because anon-elevated sample zone is in the same plane as the base, the electricfield strength cannot be varied between the base and sample zone. Byvarying the electric field strengths between the base and elevatedsample zone, optimal electric field conditions are obtained resulting inimproved desorption and ionization of the sample.

Detecting the Desorbed Ion Sample

The method of the present aspect also involves detecting the desorbedion sample 11. In an exemplary embodiment, the desorbed ion sample isdetected using an ion detector. Typically, the ion detector forms a partof a mass spectrometer.

Another embodiment is directed to a mass spectrometer apparatuscomprising an analytical assembly, an ionization source to ionize thesample, and an ion detector for detecting an ion desorbed from thesample zone. The analytical assembly comprises a chip and a conductiveelement. The chip comprises a base having a surface and one or morestructures protruding above the surface of the base. Each structurecomprises a pillar and a sample zone. The addition, the sample zonecomprises a support material. The conductive element comprises anaperture of sufficient proportion to allow passage of a molecular ionand is adapted to be at a different electrical potential than the base.

Mass spectrometers generally comprise four basic parts: a sample inletsystem, an ionization source, a mass analyzer and an ion detector (seegenerally, Kroschwitz et al., Encyclopedia of Chemical Technology, 4thed. (1995) John Wiley & Sons, New York; Siuzdak et al., MassSpectrometry for Biotechnology, (1996) Academic Press, San Diego). Massanalyzers effect separation of ions emerging from an ion source based onthe mass-to-charge ratio, m/z. A variety of mass analyzer apparatusesare useful in the current invention, including linear quadrupole (Q),time-of-flight (TOF), ion cyclotron resonance (ICR), ion traps, magneticsector and combinations and variation thereof, including tandem massspectrometers. A variety of ion detectors are usefull in the currentinvention including, for example, Faraday cups, electron multipliers,photomultiplier conversion dynodes, high energy dynode detectors, arraydetectors, Fourier transform ion cyclotron resonance detectors, and thelike.

Ionization sources are described above (see Desorption and Ionizationsection). Ionization sources include, for example, electron ionization,fast atom bombardment, laser desorption and electrospray.

FIG. 5 illustrates an exemplary method of detecting the desorbed ionsample 11 using a mass spectrometer. A laser source 10 directs laserradiation to the sample zone 6 thereby producing a desorbed ion sample11. The desorbed ion sample enters the inlet of a mass spectrometer 12that forms part of a mass spectrometer. Typically, the space within theinlet of the ion detector 12 is under a high vacuum and is, therefore,of lower pressure in relation to the space outside the inlet 12. In anexemplary embodiment, the method of desorption is MALDI and the massanalyzer it s TOF analyzer.

In an exemplary embodiment, the inlet of the ion detector 12 comprises adifferent electrical potential than the base.

Allowing the Desorbed Ion Sample to Pass Through an Aperture in aConductive Element

In an exemplary embodiment, the methods of the present inventioncomprise allowing the desorbed ion sample to pass through an aperture ina conductive element, wherein the conductive element comprises adifferent electrical potential than the base.

FIG. 6 illustrates an exemplary method comprising allowing the desorbedion sample to pass through an aperture in a conductive element. Afterdesorbing the sample 8 from the sample zone 6, the resulting desorbedion sample 11 is allowed to pass through an aperture 13 in theconductive element 14. Typically, the conductive element 14 comprises adifferent electrical potential than the base 2. For example, theconductive element can be at a potential of 60 volts and the base 2 canbe at a potential of 30,000 volts.

Elevating the sample zone with respect to the chip base provides anadvantage in allowing the desorbed ion sample to pass through anaperture in a conductive element. Because the tip is closer to theplate, the sample zone is subjected to a higher electric field than witha non-elevated sample zone. The higher electric field results in moreefficient passage of the ion through the aperture. In addition,isolation of specific samples may be more efficient because the elevatedsample zone allows the electric filed produced by the conductive elementto focus on an individual structure.

Conductive elements of the present invention comprise at least oneaperture. In One embodiment, the conductive element comprises aplurality of apertures arranged in an array format. In anotherembodiment, the conductive element comprises a single aperture.

In another embodiment, the position of the chip is translatable, therebyallowing alignment of an aperture with a structure whereby the desorbedion sample passes through the aperture. Thus, in an exemplaryembodiment, the method comprises aligning the aperture with one of thestructures whereby the desorbed ion sample passes through the aperture.Typically, the desorbed ion sample passes through the aperture beforedetection of the desorbed ion sample but after desorbing the sample fromthe chip.

In another embodiment, the conductive element is translatable, therebyallowing alignment of an aperture with a structure. In yet anotherembodiment, both the chip and the conductive element are translatable.Regardless of which component is translatable, the pillar 5 and theaperture 13 can be aligned with respect to each other.

Conductive elements of the present invention comprises a differentelectrical potential than the base. The electrical potential istypically sufficiently high to create a magnetic field of sufficientstrength to shuttle the desorbed ion sample through the aperture. Theconductive element comprises a material capable of conducting anelectrical current such as copper, aluminum and alloys thereof. Avariety of conductive materials are useful as components of a conductiveelement, such as conductive metals or semi-conductive silicon materials.

Conductive elements may be of any suitable geometry (e.g. rectangular,circular, octahedral etc.). The conductive element may be of anysuitable height and, width. In an exemplary embodiment, the conductiveelement is more than 2 cm in height. In another exemplary embodiment,the conductive element is less than 20 μm in height. In an exemplaryembodiment, the conductive element is more than 10 cm in width ordiameter. In another embodiment, the conductive element is less than 100μm in width or diameter.

Apertures of the present invention are of sufficient dimension to allowpassage of a desorbed ion sample. Thus, the size of the desorbed ionsample will determine the minimum diameter of the aperture. In anexemplary embodiment, the aperture is from about 5 angstroms to about 50angstroms in diameter. In another embodiment, the aperture is from about50 angstroms to about 500 angstroms in diameter. In another embodiment,the aperture is from about 50 nm to about 500 nm in diameter. In anotherembodiment, the aperture is from about 500 nm to about 1000 nm indiameter. In another embodiment, the aperture is from about 1 μm toabout 50 μm in diameter. In another embodiment, the aperture is fromabout 50 μm to about 500 μm in diameter. In another embodiment, theaperture is from about 500 μm to about 1000 μm in diameter. In anotherembodiment, the aperture is from about 1 to 1000 mm. In anotherembodiment, the aperture is from about 1 to 5 cm.

In another embodiment the laser source is directed to the sample zonethrough the aperture. Typically, the laser is a pulsed laser and istimed so as not to disrupt the desorbed ion samples from passing throughthe aperture.

In another embodiment, the laser radiation is directed to the samplezone through a window 15 in the conductive element 14 through an (seeFIG. 7). The window 16 is typically an aperture or a non-light absorbingmaterial such as glass or silicon-based material. The non-lightabsorbing material is typically inserted into the conductive elementafter forming a hole into which the material is inserted. The window canbe of any size suitable for allowing laser radiation to pass.

Any one or more features of any embodiment of using the chip, desorbingand ionizing the sample, detecting the desorbed ion sample, or allowingthe desorbed ion sample to pass through an aperture in a conductiveelement described above can be adapted or incorporated into an assemblyor apparatus.

For example, in one embodiment, the present invention provides ananalytical assembly comprising a chip and a conductive element. The chipcomprises a base having a surface and one or more structures protrudingabove the surface of the base. Each structure comprises a pillar and asample zone. In addition, the sample zone comprises a support material.The conductive element comprises an aperture of sufficient proportion toallow passage of a molecular ion and is adapted to be at a differentelectrical potential than the base. The pillar, base, aperture, samplezone, support, aperture and all other elements of the assembly comprisethe same properties, parameters and characteristics as described in theabove embodiments.

The Sample

A variety of samples are analyzed using the methods of the currentinvention. Samples comprise biological materials derived from a bodily,cellular, viral and/or prion source. Some samples are derived frombiological fluids such as blood and urine. In some embodiments, thebiological fluids include whole cells, cellular organelles or cellularmolecules such as a protein, protein fragment, peptide, carbohydrate ornucleic acid. The biological material can be endogenous ornon-endogenous to the source. For example, in one embodiment, thebiological material is a recombinant protein harvested from a bacteriaand engineered using molecular cloning techniques (see generally,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which isincorporated herein by reference). In another embodiment, the samplecomprises a chemically synthesized biological material such as asynthetic protein, protein fragment, peptide, carbohydrate or nucleicacid.

In some embodiments, the samples are in the form of liquids when theycontact the sample zone. When liquid samples are on the sample zone, theliquid samples may be in the form of discrete deposits. Any suitablevolume of liquid may be deposited on the sample zone. For example, theliquid samples that are deposited on the sample zones may be on theorder of about 1 microliter or less. In other embodiments, the liquidsamples on the sample zones may be on the order of about 10 nanolitersor less (e.g., 100 picoliters or less).

In yet other embodiments, liquid media need not be retained in thesample zones after liquid from a dispenser contacts the sample zone.

In another exemplary embodiment, the biological material sample may beprocessed in the sample zone by contacting the sample with a processingreagent. Processing reagents typically function to prevent the analytesin the sample zone from refolding, enhance the mass spectrometricresponse, improve the mass spectrometric fragmentation, label thesamples to improve the mass spectrometric selectivity, cleave thesample, unfold the sample, and/or derivatize the sample.

In another embodiment, the processing reagent can separate a sample thathas been covalently or noncovalently immobilized to the sample zone. Forexample, where a disulfide bond immobilizes the sample to the samplezone, the processing agent is a reducing agent (such a dithiothreitol)that disrupts the disulfide linkage and separates the sample from thesample zone. In another embodiment, the processing reagent can separatea sample from a binding reagent (see below). For example, where anantibody binding reagent immobilizes a sample to the sample zone, adenaturant (such as guanidinium hydrochloride) function to disrupt thenoncovalent bonds and separate the sample from the sample zone.

Binding Reagents

In an exemplary embodiment, the sample zone 6 comprises a surfacecoating comprising a binding reagent, wherein the binding reagentinteracts with the sample. In one embodiment, the surface coating 16coats all (see FIG. 8(a)) or a portion of the support material 9. Inanother embodiment, the surface coating coats a layer within the surfacezone that does not contain the support material.(see FIG. 8(b)). Thelayer typically is positioned above the support material at the top ofthe sample zone.

In an exemplary embodiment, binding reagent of the present invention arecovalently bound to the support material. The binding reagent may becovalently bound using a variety of covalent chemical linkages known.Useful covalent linkages may be found, for example, in texts relating,tothe art of solid phase synthesis of biomolecules such as peptides andnucleic acids (see, e.g., Eckstein et al., Oligonucleotides andAnalogues: A Practical Approach, (1991); Stewart et al., Solid PhasePeptide Synthesis, 2nd Ed., (1984))

In another embodiment, the binding reagent is non-covalently bound tothe support material. A variety of methods of non-covalently binding areuseful in the present invention and include, for example, methods basedon ionic interactions, hydrogen bonding, hydrophobic interactions,hydrophilic interactions and hydrogen bonding interactions.

In an exemplary embodiment, the interaction between the binding reagentand the sample is a specific binding event. In a specific binding event,the binding reagent has a high affinity to a specific element of thesample. In an exemplary embodiment, the sample comprises a protein andthe binding reagent is an antibody molecule that has a high affinity toa specific site of the protein. In another exemplary embodiment, thesample comprises a nucleic acid and the binding reagent is a nucleicacid capable of specifically hybridizing with the sample nucleic acid.In another exemplary embodiment, the sample comprises a nucleic acidbinding protein and the binding reagent comprises a nucleic acid capableof specifically binding to the nucleic acid binding protein.

Binding reagents function to bind the sample to the sample zone. Thebinding reagent may bind to the sample zone and substantially all of theliquid medium may be removed from the sample zone, leaving only thecapture agent at the sample zone. A variety of binding reagents arecapable of binding the samples of the invention to the sample zone.

Suitable binding reagents may be organic or inorganic in nature, and maybe biological molecules such as proteins, polypeptides, DNA, RNA, mRNA,antibodies, antigens, etc. Other suitable analytes may be chemicalcompounds that may be potential candidate drugs. Reactants may includereagents that can react with other components on the sample zones.Suitable reagents may include biological or chemical entities that canprocess components at the sample zones. For instance, a reagent may bean enzyme or other substance that can unfold, cleave, or derivatize theproteins at the sample zone. Suitable liquid media include solutionssuch as buffers (e.g., acidic, neutral, basic), water, organic solvents,etc. Binding reagents are well known in the art and include, but are notlimited to, glutathione-S-transferase (GST), maltose-binding domain,chitinase (e.g. chitin binding domain), cellulase (cellulose bindingdomain), thioredoxin, protein G, protein A, T7 tag, S tag, Histidine-6,protein kinase inhibitor, HA, c-Myc, trx, Hsc, Dsb, and the like.

In another exemplary embodiment, the surface coating is a thin filmcomprising a binding reagent wherein the binding reagent comprises anorganic molecule. The thin film is typically less than about 20nanometers thick. Preferably, the organic thin film is in the form of amonolayer. A “monolayer” is a layer of molecules that is one moleculethick. In some embodiments, the molecules in the monolayer may beoriented perpendicular, or at an angle with respect to the surface towhich the molecules are bound. The monolayer may resemble a “carpet” ofmolecules. The molecules in the monolayer may be relatively denselypacked so that proteins that are above the monolayer do not contact thelayer underneath the monolayer. Packing the molecules together in amonolayer decreases the likelihood that proteins above the monolayerwill pass through the monolayer and contact a solid surface of thesample structure.

In another embodiment, the binding reagent comprises an affinity tag. Anaffinity tag is a functional moiety capable of directly or indirectlyimmobilizing a component such as a protein. The affinity tag may includea polypeptide that has a functional group that reacts with anotherfunctional group on a molecule in the organic thin film. Suitableaffinity tags include avidin and streptavidin.

In another embodiment, the surface coating further comprises an“adaptor” that directly or indirectly links a binding reagent to apillar. In some embodiments, an adaptor may provide an indirect ordirect link between an affinity tag and a capture agent.

Other examples of surface coatings and binding reagents are described inU.S. patent application Ser. Nos. 09/115,455, 09/353,215, and09/353,555, and U.S. Pat. No. 6,454,924, which are herein incorporatedby reference in their entirety for all purposes, and are assigned to thesame assignee as the present application. These U.S. patent applicationsdescribe various layered structures that can be on the pillars inembodiments of the invention.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding equivalents of thefeatures shown and described, or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention claimed. Moreover, any one or more features of any embodimentof the invention may be combined with any one or more other features ofany other embodiment of the invention, without departing from the scopeof the invention. For example, any feature of the methods of analyzing asample described above can be incorporated into any of the assemblies,chips, or systems without departing from the scope of the invention.

In addition, the patents and scientific references cited herein areincorporated by reference in their entirety.

1. A method comprising: (a) desorbing a sample from a chip to produce adesorbed ion sample, wherein the chip comprises: i. a base having asurface, and ii. one or more structures protruding above the surface ofthe base, each structure comprising a pillar and a sample zone, whereinthe sample zone comprises a support material and the sample; (b)detecting the desorbed ion sample with an ion detector.
 2. The method ofclaim 1 further comprising allowing the desorbed ion sample to passthrough an aperture in a conductive element, wherein the conductiveelement comprises a different electrical potential than the base.
 3. Themethod of claim 2, wherein the position of the chip is translatable,wherein the method further comprises aligning the aperture with one ofthe structures whereby the desorbed ion sample passes through theaperture after (a) but before (b).
 4. The method of claim 1, whereinsaid support material receives radiation.
 5. The method of claim 1,wherein each pillar and the base comprise the support material thatreceives radiation.
 6. The method of claim 1, wherein said supportmaterial is porous.
 7. The method of claim 1, wherein said supportmaterial is conducting or semiconducting.
 8. The method of claim 1,wherein said support material is capable of transferring energy to thesample after receiving radiation.
 9. The method of claim 1, wherein thesupport material is coated with a surface coating comprising a bindingreagent, wherein the binding reagent interacts with the sample.
 10. Themethod of claim 9, wherein the interaction between the binding reagentand the sample is a specific binding event.
 11. The method of claim 1,wherein the pillar and sample zone are identical in chemicalcomposition.
 12. The method of claim 1, further comprising directingradiation at the sample zone before (a).
 13. The method of claim 2,further comprising directing radiation at the sample zone before (a)through a window in the conductive element.
 14. The method of claim 1,wherein the ion detector forms part of a mass spectrometer.
 15. Ananalytical assembly comprising: a. a chip comprising: i. a base having asurface; and ii. one or more structures protruding above the surface ofthe base, each structure comprising a pillar and a sample zone, whereinthe sample zone comprises a support material; and b. a conductiveelement comprising: i. an aperture of sufficient proportion to allowpassage of a molecular ion; and ii. is adapted to be at a differentelectrical potential than the base.
 16. The analytical assembly of claim15, wherein said support material is adapted to receive radiation. 17.The analytical assembly of claim 16, wherein each pillar and the basecomprise said support material.
 18. The analytical assembly of claim 15,wherein said support material is porous.
 19. The analytical assembly ofclaim 15, wherein said support material is conducting orsemi-conducting.
 20. The analytical assembly of claim 15, wherein saidsupport material is capable of transferring energy to a sample afterreceiving radiation.
 21. The analytical assembly of claim 15, whereinthe position of the chip is translatable, thereby allowing alignment ofthe aperture with a structure whereby a sample desorbed from thestructure and attracted toward the conductive element passes through theaperture.
 22. The analytical assembly of claim 15, wherein the samplezone is coated with a surface coating comprising a binding reagent,wherein the binding reagent interacts with a sample.
 23. The analyticalassembly of claim 22, wherein the interaction between the bindingreagent and the sample is a specific binding event.
 24. The analyticalassembly of claim 15, wherein the pillar and sample zone are identicalin chemical composition.
 25. The analytical assembly of claim 15,wherein the conductive element further comprises an ion detector. 26.The analytical assembly of claim 15, wherein the conductive elementfurther window, wherein radiation is passed through said window.
 27. Amass spectrometer apparatus comprising: (a) an analytical assemblycomprising (i) a chip comprising: A. a base having a surface; and B. oneor more structures protruding above the surface of the base, eachstructure comprising a pillar and a sample zone, wherein the sample zonecomprises a support material; and (ii) a conductive element comprising:A. an aperture of sufficient proportion to allow passage of a molecularion; and B. is adapted to be at a different, electrical potential thanthe base. (b) an ionization source to ionize the sample; and (c) an iondetector for detecting an ion desorbed from the sample zone.